Our objective is to determine how cytoplasmic microtubules and contractile proteins interact during mitosis and other cellular movements. We will use morphological and biochemical methods to investigate the idea that microtubules act as a scaffolding for the contractile proteins, actin and myosin, which actually generate the forces for movement. We will determine the relative positions of actin, myosin and microtubules in the mitotic apparatus at about 0.5 micrograms resolution using fluorescence microscopy of cells stained with pairs specific ligands conjugated with contrasting fluorochromes. Antibodies which bind specifically to myosin or tubulin and heavy meromyosin, which binds specifically to actin filaments, will be conjugated with rhodamine or fluorescein and used to double stain cultured cells fixed at different stages of mitosis. Microspectrofluorometry of cells stained with rhodamine-anti-myosin will provide an estimate of the myosin of the mitotic spindle in situ. A second morphological project will be to examine the interactions of actin filaments, myosin and microtubules in the mitotic spindle at the ultrastructural level. Microtubules are adequately preserved by routine procedures, and we are developing new methods to properly fix actin filaments, so that both of these fiber systems can be visualized in routine thin sections. Myosin filaments are impossible to identify in routine thin sections, so we will use antimyosin conjugated with an electron dense marker to localize myosin in dividing cultured cells lysed under conditions which stabilize the mitotic spindle. At the same time we will localize myosin in the cytokinetic contractile ring. Biochemical studies are planned to search for, characterize and determine the significance of interactions between microtubules and purified actin or myosin. In the event that no such interactions are found, we will examine the "brain dynein" as possible energy transducing enzyme linked directly to cytoplasmic microtubules.